Biochemistry 1992,31, 6193-6202
6193
Cloning and Characterization of the Casein Kinase I1 a! Subunit Gene from the Lymphocyte-Transforming Intracellular Protozoan Parasite Theileria parvat,$ Onesmo K. ole-MoiYoi,* Chihiro Sugimoto,%Patricia A. Conrad,” and Michael D. Macklin International Laboratory for Research on Animal Diseases, P.O.Box 30709, Nairobi, Kenya Received January 24, 1992; Revised Manuscript Received April 21, 1992 ABSTRACT: Theileria parua is an obligate intracellular protozoan parasite which is the causative agent of
East Coast fever, an acute, leukemia-like disease of cattle. The intralymphocytic stage of the parasite induces blastogenesis and clonal expansion of quiescent bovid lymphocytes. Experiments in our laboratory have shown a marked increase of casein kinase II- (CK II-) like activity in T . parua-transformed lymphocytes. We have also detected CK I1 activity in purified T . parua schizonts. To explore the significance of this increase, we used a Drosophila melanogaster CK I1 a cDNA probe [Saxena et al. (1987) Mol. Cell Biol. 7 , 3409-34171 to isolate a T . parua genomic clone encoding a CK I1 catalytic subunit. The clone contains 50 200). Northern blot a 1.3-kb open reading frame coding for a predicted protein of 420 amino acids (MI analysis revealed a single transcript of 1.65 kb. The deduced T . parua CK I1 catalytic subunit sequence shows, over 321 residues comprising the C-terminus of the molecule, extensive identity with CK I1 a and a’ sequences from both vertebrate and invertebrate organisms. The T . parva CK I1 subunit amino acid sequence displays 68% identity with the Drosophila a subunit and 67% with the Caenorhabditis elegans a subunit but only 58% and 56% sequence identity with the Saccharomyces cerevisiae a and a‘ subunits, respectively. Comparison of the T . parua sequence with higher eukaryotic a and a’sequences reveals that it is most identical with the a subunit. A unique component of the T. parua CK I1 a subunit is a 99 amino acid sequence at the N-terminus, which contains a sequence motif with features characteristic of signal peptides. C a s e i n kinase I1 is a serine/threonine protein kinase that is messenger-independent and that has, as its potential physiologic substrates, a diverse and expansive spectrum of proteins involved in metabolism, differentiation, and cellular proliferation [for recent reviews see Edelman et al. (1987), Krebs et al. (1988), and Tuazon and Traugh (1991)l. Casein kinase (CK) I1 is ubiquitous among eukaryotes and the enzyme or the genes encoding its subunits have been isolated from many sources, including mammalian and avian species (Hathaway & Traugh, 1982; Maridor et al., 1991), Drosophila (Glover et al., 1983; Saxena et al., 1987), yeast (Meggio et al., 1986; Padmanabha & Glover, 1987; Chen-Wu et al., 1988; Padmanabha et al., 1990), Caenorhabditis elegans (Hu & Rubin, 1990) and Dictyostelium discoideum (Renart et al., 1984). The enzyme has been shown to be predominantly cytosolic in distribution but has also been localized to a variety of subcellular organelles, including the nuclear compartment (Hathaway & Traugh, 1983), coated pits (Bar-Zvi & Branton, 1986) and mitochondria (Damuni & Reed, 1988). Furthermore, immunocytochemical studies have shown a marked shift of CK I1 from the cytoplasm into the nucleus during active proliferation of bovine adrenocortical cells in culture (Filhol et al., 1990). Employing CK I1 subunit-specific antibodies in immunocytochemical studies, Yu et al. (1991) showed that, in HeLa cells, CK I1 a and 6 subunits are predominantly cytoplasmic during interphase but become distributed throughout the cell during mitosis. In contrast, the a’subunit is mostly nuclear in localization during G1 but moves to the ‘This is ILRAD publication no. 1034. *Thenucleic acid sequence in this paper has been submitted to GenBank under Accession Number M92048. * Corresponding author. I Present address: Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan. 11 Present address: Department of Veterinary Microbiology and Immunology, University of California at Davis, Davis, California 9561 6.
cytoplasm during ’s phase. Although these studies indicate that the localization of CK I1 is not static but rather depends on the stage of the cell cycle, the report by Krek et al. (1992) suggests that, in primary chick embryo fibroblasts, chicken hepatoma cells, and HeLa cells transfected with cDNAs encoding the subunits of chicken CK 11, the enzyme is predominantly localized in the nucleus. In most higher eukaryotes, the type I1 casein kinases are heterotetrameric, with possible holoenzyme isoforms of ad2, ( Y C Y ’ ~ and ~, a’d2[for review see Tuazon and Traugh (1991)l. The a subunits (MI 37000-44000) have been shown to be catalytic, while the fl subunit (MI24000-28 000), which undergoes autophosphorylationin the presence of ATP or GTP, has a regulatory role (Hathaway et al., 1981; Feige et al., 1983; Cochet & Chambaz, 1983; Meggio & Pinna, 1984; Palen & Traugh, 1991). Until recently the structural relationship between the catalytic subunits of CK I1 was unclear (Dahmus et al., 1984). However, now with the availability of CK I1 sequences of these subunits, evidence has been presented showing that, in several species, the a and a’ subunits are products of different genes (Chen-Wu et al., 1988; Meisner et al., 1989; Padmanabha & Glover, 1987; Padmanabha et al., 1990; Lozeman et al., 1990; Litchfield et al., 1990; Maridor et al., 1991). However, in Drosophila only one form of the catalytic subunit has been reported (Saxena et al., 1987). Casein kinase I1 phosphorylates serine or threonine residues in a large number of potential protein substrates, including a variety of enzymes, cell membrane receptors, cytoskeletal proteins, transcription factors, and products of oncogenes [Meggio et al., 1984; see review by Tuazon and Traugh (1991)l. The phosphorylated residues are usually N-terminal to a cluster of acidic amino acid residues. An increasing number of these proteins have been shown to be phosphorylated in vivo at the phosphorylation site motifs specific for CK I1 (Krebs et al., 1988; Lozeman et al., 1990). Among these are eukaryotic or viral proteins that may be involved in cell
0006-2960/92/043 1-6193$03.00/0 0 1992 American Chemical Society
6194 Biochemistry, Vol. 31, No. 27, 1992 growth or transformation including SV40 large T antigen (Krebs et al., 1988; Grasser et al., 1988), Myc (Luscher et al., 1989), DNA topoisomerase I1 (Ackerman et al., 1988), human papilloma virus E7 protein (Firzlaff et al., 1989), mouse p53 (Meek et al., 1990), and ornithine decarboxylase, an enzyme which is rapidly induced following stimulation of cells with growth-promoting agents (Meggio et al., 1987; Rosenberg-Hasson et al., 1991). Casein kinase 11 activity is enhanced within minutes after the addition of insulin to cells propagated in vitro (Sommercorn et al., 1987; Klarlund & Czech, 1988). The enzyme is also induced during cell differentiation and by exposure of cells to growth factors, such as EGF or serum (Sommercorn & Krebs, 1987; Sommercorn et al., 1987; Krebs et al., 1988; Carroll & Marshak, 1989; Ackerman & Osheroff, 1989). High levels of casein kinase I1 activity have also been reported during early developmental stages of C.elegans (Hu & Rubin, 1990) and in early chicken embryos (Maridor et al., 1991). These data suggest that casein kinase I1 may function as an important element of postreceptor, signal-transducing pathways activated by hormones and by a variety of growth factors. We have been studying the molecular mechanisms responsible for bovine T lymphocyte transformation induced by Theileria parva. T. parva, an obligate intracellular protozoan parasite transmitted by a tick vector, causes East Coast fever, an acute and often fatal lymphoproliferative disease of cattle in eastern and central Africa. The mammalian-infective stage of Theileria is the sporozoite, which is injected into the host with the saliva during feeding. Although T. parva sporozoites can enter a variety of bovine leukocytes engaging a receptor/ligand-like endocytotic process, the parasite appears to survive in, and induce proliferation of, mostly T cells (Fawcett et al., 1982; Baldwin et al., 1988). The sporozoite, which is encapsulated by the host cell plasma membrane during entry, induces rapid dissolution of that membrane soon after entry into the lymphocyte (Fawcett et al., 1982). The dissolution of the host plasma membrane surrounding the sporozoite occurs concurrently with the diminution of the electron-dense material contained in the rhoptries and micronemes, subcellular organelles which have been shown to mediate host cell entry by related parasitic protozoa such as Plasmodium spp. Within the lymphocyte, the sporozoite, whose plasma membrane is entirely exposed to the host cell cytoplasm, then differentiates into a multinucleate schizont, which induces clonal expansion and immortalization of T. pama-infected cells in vitro as well as in vivo (Baldwin et al., 1988; Emery et al., 1988). Studies on merogony in T. parva suggest that the schizont undergoes fission to form merozoites, which bud off from the lymphocyte to enter the circulation where they infect erythrocytes, within which they differentiate into piroplasms. Theileria-infected cells have been considered transformed because they exhibit cellular pleiomorphism, have short (16-25 h in vitro) doubling times and surface phenotype alterations involving differentiation antigens, and, when inoculated into athymic mice, form tumorlike masses, which metastasize and infiltrate most organs (Irvin et al., 1975; Baldwin et al., 1988; Conrad et al., 1989). The T. pama-transformed lymphocytes are thought to be primarily responsible for the pathology associated with the disease. In an attempt to determine the molecular mechanisms responsible for the T.parva-induced cellular transformation, we have found a marked increase in protein kinase activity in protein preparations from infected cells (ole-MoiYoi et al., 1990). On the basis of substrate specificity and susceptibility to various inhibitors, the enzymes we have found in T . pur-
ole-MoiYoi et al. vu-infected cells are serine/threonine kinases with the specificities of casein kinase 11. We have used Drosophila cDNA clones encoding the casein kinase I1 a or 0 subunits as probes on Southern blots of both bovine and T. parva DNA and have subsequently cloned the gene for an a subunit of the parasite enzyme. In this paper we report the isolation and characterization of the gene for T . parva casein kinase I1 a subunit. MATERIALS AND METHODS Parasites. Infection of cattle with sporozoite stabilates of different stocks of T. parva was carried out as previously described by Conrad et al. (1987) and Dolan et al. (1984). A T. parua stabilate, isolated at Muguga (TpM: ILRAD isolate 836), Kenya, was used as a source of T. parva materials described in this paper. Cloning of Bovine Lymphocytes and Infection with T. parva. IL 2-dependent bovine cell lines (B657.G6 and A19) had been cloned by limiting dilution from concanavalin A-stimulated bovine lymphocytes as previously described (Brown & Grab, 1985; Morrison et al., 1987); these cell lines belong to the CD8' subset of T cells (Baldwin et al., 1988). Infection of the IL 2-dependent cell lines with T. parva was achieved by incubating the cells with sporozoitesof T. parva purified from tick salivary gland homogenates (Dobbelaere et al., 1984). The IL 2-dependent lymphocyte clone and the same clone after infection with T. parva shall hereafter be referred to as G6 and G6TpM, respectively. The G6 and the infected G6TpM cell lines were cultured in RPMI 1640 medium containing 25 mM HEPES (Gibco Europe, Paisley, UK) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 2 mM L-glutamine, 50 pM 2-mercaptoethanol, and 50 pg of gentamycin/mL. The cells were incubated at 37 "C in an atmosphere of 3 4 % CO, in air as previously described (Brown & Grab, 1985). At times, G6TpM was also cultured in L-15 medium, which had been supplemented with 10% tryptose phosphate broth and fetal bovine serum, L-glutamine, 2mercaptoethanol, and gentamycin at the concentrations given above. Preparation of Lymphocyte Homogenates and Purification of Schizonts. G6 and G6TpM cells, in log-phase growth, were washed several times with phosphate-buffered saline (PBS, pH 7.4) and used for preparation of homogenates for protein kinase assays or for purification of schizonts from T. parvainfected lymphocytes. The cells were suspended in cold homogenization buffer (250 mM sucrose, 25 m M Tris-HC1, pH 7.2,O.OI mM EDTA, 5 mM MgCl,, 120 mM KCl, 1 mM PMSF, and 8 pg of aprotinin/mL) at a concentration of 1 X lo8 cells/mL and sonicated at 4 "C for 2 min (0.5s pulses) at a setting of 230 W with a Branson ultrasonic cell disrupter equipped with a jacket horn. The homogenates were separated into pellet and supernatant by centrifugation at 1OOOOOg for 30 min at 4 O C . The pellet was recovered and resuspended in a volume of cold homogenization buffet equal to that of the 1OOOOOg supernatant. Both the pellet and supernatant fractions were analyzed for protein kinase activity on endogenous or exogenous substrates as described below. In both cell lines, most of the enzymatic activity (>90%) was in the lOOOOOg pellets. Homogenates of schizonts purified from infected lymphocytes, as described by Sugimoto et al. (1988), were also prepared and used as a source of protein kinase. Briefly, T. parva-infected lymphocytes were suspended (4 X IO7 cells/ mL) in 10 mM HEPES, pH 7.4,150 mM NaCl, 20 mM KCl, and 1 mM CaCl, containing 20 pg/mL each of the following protease inhibitors: leupeptin, antipain, chymostatin, and truns-epoxysuccinyl-~-leucylamido-(4-guanidino) butane (E-64)
Theileria puma Casein Kinase I1 a Subunit and 2% (w/v) FicollW (Phannacia Fine Chemicals, Uppsala, Sweden). To release schizonts, the T. pama-infected lymphocytes were lysed by the addition of the cytotoxins aerolysin (10 pg/mL) or Ah-1 hemolysin (23 pg/mL), both purified from Aeromonas hydrophila. The cells were incubated at 23 OC for 10 min (aerolysin) or 15 min (Ah-1 hemolysin). Schizont aggregation was inhibited by addition of EDTA to a final concentration of 5 mM. Percoll stock, made in the HEPES-EDTA buffer, was added to the cell lysate to give a final concentration of 64.6% (v/v). The schizonts were then separated from the cellular debris by centrifugation (1 1OOOOg) through a discontinuous Percoll gradient (4564.6%) for 30 min at 4 OC. Schizonts purified in this way were essentially free from host cell debris and retained their intralymphocytic morphology as assessed by electron microscopical examination. When the analysis of phosphorylation of exogenous substrates was performed, highly diluted schizont homogenates (20-60 ng of protein) were used as a source of enzyme. For the experiments reported herein, protein kinase assays were performed by incubation, for 10-15 min at 23 OC, of reaction mixtures containing 10 mM MgC12, 120 mM KC1, 50 mM Tris-HC1 (pH 7.2), 1 mM 3-isobutyl-l-methylxanthiie (IBMX), 15 pM aprotinin, 40 pM [T-~~P]ATP (specific activity 7500 cpm/pmol) and 20-80 pg of protein as substrate. To this mixture was added 20-60 ng of schizont lysate as enzyme source. Polyacrylamide Gel Electrophoresis. Samples, each containing 20-60 pg of protein, were incubated in vitro in the protein kinase assays described above and, after incubation, boiled in sodium dodecyl sulfate (SDS) sample buffer for 5 min and run on SDS-polyacrylamide gradient (5-1 7.5% or 7.5-15%) gels according to Laemmli (1970). The gels were stained with Coomassie blue, destained, and dried. The dried gels were exposed to Fuji RX 100 film for autoradiography, mostly at -80 OC with intensifying screens for varying periods of time. For better resolution of phosphorylated protein bands, it was occasionally necessary to carry out the exposure at 23 OC using Hyperfilm 0 max (Amersham International plc., Amersham, UK) without intensifying screens. DNA Preparation. DNA was prepared from the G6 and G6TpM lymphocyte clones as well as from T. pama piroplasms according to Conrad et al. (1987). Briefly, purified piroplasms or lymphoblastoid cell pellets (final concentration 5 X lo7 cells/mL) were suspended in 1X TNE (25 mM Tris-HC1, 5 mM EDTA, and 100 mM NaC1, pH 8.0), and SDS and boiled RNase A were added to the cell suspensions to give final concentrations of 0.5% and 100 pg/mL, respectively. The cell lysates were mixed gently and incubated at 37 OC for 1-2 h. Proteinase K (BRL, Gaithersburg, MD) was added to a final concentration of 100 pg/mL and the preparations were incubated for 3 h at 50 OC. Further extraction of DNA was performed as described by Sambrook et al. (1989). Genomic Library Screening. A library of T. puma (Muguga) piroplasm sheared genomic DNA was constructed in Xgtll as previously described (Sambrook et al., 1989; Conrad et al., 1987). The library, which yielded 1.1 X lo6independent recombinant phage clones from 0.1 pg of T. puma DNA, was plated out at 5 X lo3plaque-forming units (pfu) per 150-mm plate and screened using a cDNA clone of D. melanogaster CK I1 a subunit (Dm 95, a gift from Dr. Claiborne V. C. Glover; Saxena et al., 1987). After incubation, duplicate filters (Schleicher 8c Schiiell, Keene, NH) were prepared, the DNA was denatured, and the filters were neutralized and baked at 80 "C for 2 h. The Drosophila CK I1 a cDNA probe was
Biochemistry, Vol. 31, No. 27, 1992 6195 labeled by random priming according to Feinberg and Vogelstein (1983) to a specific activity of about 1 X lo9 cpm/pg of DNA and used to probe the duplicate filters in 4X SSC (1X SSC is 0.15 M NaCl and 0.015 M sodium citrate), 1OX Denhardt's solution, 0.1% SDS, and 0.1% sodium orthophosphate at 50 OC for 16 h. After hybridization, the filters were washed twice in 4X SSC/O.l% SDS at 50 OC for 1 h and once in 2X SSC/O.l% SDS at 50 OC for 1 h. The filters were then dried and exposed for 16 h to Fuji RX 100 f h (Fuji Photo Film Co., Japan) with intensifying screens at -80 OC. One positive clone (XgTpMCK2) was identified and was plaque-purified by two additional rounds of screening using the conditions described above. This clone contained a 4.5-kb insert of T. puma DNA. Subcloning and DNA Sequencing. The 4.5-kb genomic DNA fragment of T. puma in the clone XgTpMCK2 was subcloned into pUC19 and a partial restriction map was generated. Subsequently, a 1.4-kb EcoRI/HpaII fragment and a 0.9-kb HpaII fragment were also subcloned into pUC19. Nested deletions were generated by digestion of the DNA inserts with Bal31 exonuclease. The inserts were transferred into M13mp18/19 and all of the sequencing was performed using the dideoxy chain-termination method (Yanisch-Perron et al., 1985; Sanger et al., 1977), predominantly using Sequenase (US. Biochemicals, Cleveland, OH) and [cY-~'S]~ATP according to the manufacturer's instructions. The sequencing gels were read using a sonic digitizer interfaced to a microcomputer (Science Accessories Corp.). Each base was sequenced at least twice on each of the strands. The sequence data were analyzed by DNASIS/PROSIS (Hitachi Software Engineering, Brisbane, CA) on an IBM PS/2 Model 80 computer. Amino acid sequence comparisons were performed using the Clustal software program (Higgins & Sharp, 1988). Southern Blot Analysis. DNA prepared from uninfected or T. pama-infected lymphocytes or from piroplasms was digested with various restriction enzymes and fractionated on 0.8% agarose gels, which were subsequently processed as described by Sambrook et al. (1989). DNA fragments were transferred onto Hybond N+ nylon filters (Amersham International) and hybridized with either 32P-labeled Dm 95 Drosophila cDNA (Saxena et al., 1987) or with an Eco0109I/XhoI fragment of T. puma DNA from the XgTpMCK2 clone. The 888-bp Eco0109I/XhoI fragment contained most of the open reading frame of the T. puma CK I1 gene. In addition to the low and intermediate stringency washing conditions described above, Southern blot filters hybridized with the latter probe were additionally washed in 0.1X SSC/OS% SDS at 65 OC for 1 h. RNA Isolation and Northern Blot Analysis. Total RNA was prepared as described by Han et al. (1987) from the G6 lymphoblasts in log-phase growth, the from G6TpM cell line, and from T. puma piroplasms. Poly(A)-enriched RNA was isolated by oligo(dT)-cellulose (Type 3) chromatography according to the manufacturer's instructions (Collaborative Research Inc., Bedford, MA). Poly(A)-rich RNA was denatured, fractionated on l .4% agarose-formaldehyde gels, and transferred onto Hybond N+ nylon membranes (Amersham). Antisense 32P-labeledRNA transcripts were synthesized in vitro using a 1.5-kb T. puma CK I1 a cDNA cloned into pBluescript SK (Stratagene, La Jolla, CA). Hybridization was performed for 16 h at 60 "C according to the instructions provided by the supplier of T7 RNA polymerase (Stratagene). After hybridization, the filters were washed twice, each time for 30 min, in 0.5X SSC/OS% SDS at 65 "C. Autoradiography was performed at -80 OC as described above.
6196 Biochemistry, Vol. 31, No. 27, 1992 Construction and Screening of a cDNA Library. Doublestranded cDNA was made from poly(A)-enriched RNA (5 pg), prepared from T. parva-infected bovine lymphocytes, according to the instructions provided by the manufacturer (BRL cDNA Synthesis System). The first strand was primed with oligo(dT) and the second strand was made with RNase H and DNA polymerase I. The double-stranded cDNA was methylated on EcoRI sites and blunt-end ligated to EcoRI linkers (New England Biolabs, Beverly, MA) using T4 DNA ligase (Amersham). The EcoRI linkers were removed by digestion with EcoRI and separated from the cDNA by size fractionation on a 5-20% potassium acetate gradient in a Beckman SW50.1 rotor for 3 h at 49000 rpm at 22 "C (Aruffo & Seed, 1987). The cDNA with sizes greater than 0.8 kb was mixed with Xgtl 1 arms (Promega, Madison, WI) and ligated using T4 DNA ligase. The library had about 2 X lo6 independent recombinants. This library was screened using the 32P-labeledEcoO109I/XhoI fragment as probe, employing the high-stringency washing conditions described above. Several cDNA clones were isolated and the clone with the largest insert (1.5 kb) was further characterized. RESULTS Identification of Casein Kinase 11Activity in T. parva Schizonts. Comparison of kinase activity of 1OOOOOg particulate or supernatant preparations from the IL 2-dependent G6 clone and those from the G6TpM cell line revealed that the infected cell line had an 11-18-fold increase in kinase activity per microgram of protein (ole-MoiYoi et al., 1990). In order to understand the significance of this finding, it was essential to determine the contribution of the intralymphocytic schizont to the total protein kinase activity detected in the T. pama-infected cells. For these studies, we employed homogenates prepared from schizonts purified from infected lymphocytes as an enzyme source to phosphorylate heat-inactivated lOOOOOg pellet preparations from the infected lymphocytes as well as a variety of commercially-available kinase substrates. These experiments revealed that T. parua schizonts had a kinase activity that was capable of phosphorylating caseins, phosvitin, and glycogen synthase but that failed to phosphorylate, to any extent, histones 2A, 111-S, or V-S, protamine, or kemptide (data not shown). Figure 1 depicts an autoradiogram of an SDS-polyacrylamide gradient (7.5-15%) gel in which substrates were resolved after phosphorylation with the schizont-associated kinase. The phosphorylation of mixed casein or phosvitin was essentially abolished by 750 ng of heparin/mL (Figure 1, compare lanes 2 and 3 with lanes 5 and 6, respectively), whereas that of glycogen synthase was less affected (Figure 1, compare lane 4 with lane 7). There were only a few faint bands ( M , 86000 and 14000-17000) visible in the schizont lysate itself (Figure 1, lane 8). There were no detectable protein bands in the M, range of bovine CK I1 subunits. The schizont lysate nevertheless phosphorylated the heat-inactivated (94 "C, 10 min) lOOOOOg pellet proteins, which by themselves had no residual kinase activity, to give patterns (Figure 1, lane 1) similar to those seen when uninactivated endogenous enzymatic activity was used to phosphorylate endogenous lymphocyte (G6TpM) substrates (ole-MoiYoiet al., manuscript in preparation). The enzyme in the schizont preparations was activated by spermine and polylysine but not by cyclic nucleotides (data not shown). Cloning of a T. parva CK 11Gene. The preliminary experiments described above suggested that T. parva schizonts had a casein kinase 11-like enzyme. We therefore used the Drosophila CK I1 a! or @ cDNAs (Dm 95 and Dm 98, respectively; Saxena et al., 1987) to probe Southern blots of
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FIGURE1 : Inhibition by heparin of protein phosphorylation by a T. puma schizont-associated CK 11. A homogenate of a highly purified schizont preparation (lane 8) was used to phosphorylate 20 pg each of mixed casein, phosvitin, and glycogen synthase (lanes 2,3, and 4, respectively). For each substrate, a reaction mixture containing heparin (750 ng/mL) was processed in parallel (depicted in lanes 5-7). Phosphorylation of heat-inactivated (94 "C,10 min) lOOOOOgprotein pellet from T. pamu-infected lymphocytes, which had no detectable residual kinase activity, was also used as substrate for the schizont-associated kinase (lane 1). The arrow depicts a protein band (M,86000) present in both the schizont and lymphocyte pellet preparations. Molecular masses of the markers (m) are in kilodaltons.
DNA prepared from the uninfected IL 2-dependent G6 lymphocyte clone, G6TpM, and T. parva piroplasm DNA. The Drosophila CK I1 @ cDNA hybridized to bovine DNA, but not to Theileria DNA under intermediate stringency (2X SSC, 50 "C) washing conditions. In contrast, the Drosophila CK I1 a! cDNA probe showed no hybridization to bovine DNA at washings of intermediate stringency but did hybridize to fragments of approximately 15 kb and 10 kb of EcoRI- and HindIII-digested T. parua DNA, respectively (data not shown; however, see below). When the Drosophila CK I1 CY cDNA probe was used to screen a library of T.parva piroplasm genomic DNA in X g t l 1, one positive clone, containing a 4.5-kb insert, was identified when the filters were washed at the same stringency as for Southem blots. This clone, XgTpMCK2, was plaque-purified and used for preparation of DNA. A 1.4-kb EcoRIIHpaII fragment and a 0.9-kb HpaII fragment were then subcloned for sequencing. Bal3 1 nuclease digestion time points of these two fragments were generated for cloning into M 13mpl8/ 19 for sequencing. A partial restriction map of the insert cloned in XgTpMCK2 is shown in Figure 2A. Properties of the CK 11Gene Sequence of T. parva. The stop codon map for the three reading frames of the cloned sequence in the forward direction revealed an open reading frame (reading frame 1) with an in-frame TGA stop d o n 25 nucleotides upstream of a potential ATG start d o n (Figure 2B). The open reading frame (ORF) starts from nucleotide 50 and extends to a TAA termination codon ending at nucleotide 1336. Within the 5' part of the T. parva CK I1 a! gene encoding the 99 amino acid sequence unique to this organism, there were three potential ATG initiation codons starting at nucleotides 74,290, and 299. We have not found any typical consensus sequences for translation initiation within the expected distance from any of these ATG initiation codons. The ORF encodes a protein of 420 amino acids with a calculated M,of 50200. The translated region of this gene is 64.7% AT, whereas the 3' untranslated region is 80.4% AT. However, the short 5' untranslated region is only slightly more AT-rich (65.3%) than the coding region. There was a potential polyadenylation signal (AATAAA) starting at nucleotide 1368. Interestingly, sequence analysis of two cDNA clones
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Biochemistry, Vol. 31, No. 27, 1992 6197
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FIGURE2: Sequence of the T.parua CK I1 cy subunit gene. (A) Schematic representation and restriction map of the 4.5-kb T.parua piroplasm
genomic DNA fragment cloned in the recombinant phage XgTpMCK2. The hatched boxes represent the noncoding regions (UTR) of the gene. The open reading frame is shown by the open box. The sequencing strategy is depicted by the arrows under the diagram. Each base was sequenced over at least twice on each strand. (B) Nucleotide and deduced amino acid sequence (single-letter code) of the only major open reading frame within the sequenced fragment of the XgTpMCK2 clone. Numbers at the left and right margins indicate p i t i o n s of nucleotides and amino acids, respectively. A portion of the putative signal sequence (amino acid residues 9-26) is underlined. When the deduced sequence was analyzed using the weight-matrix scanning algorithm described by von Heijne (1986), a part of the underlined sequence was identified as a potential signal peptide. Potential N-linked glycosylation sites are boxed. The ‘transcript-instability” sequence motifs in the 3’ UTR are overlined. The two stop codons discussed in the text are shown as * (5’) and ** (3’).
coding for the T.puma CK I1 CY subunit revealed that identity between the genomic sequence and the cDNA sequences ended at nucleotide 1374, one nucleotide beyond the potential poly(A)
addition signal. This was immediately followed by a poly(A) tail. Such short (3-6 nt) nucleotide sequences between potential polyadenylation signals and the poly(A) tails have been
6198 Biochemistry, Vol. 31, No. 27, 1992 T . parvd 1
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FIGURE3: Comparison of the T.parva CK I1 cy sequence with casein kinase I1 catalytic subunits from other invertebrates. The deduced amino acid sequence of T. parva CK-I1 cy is aligned with other CK I1 sequences from S. cereuisiae cy (CKAl; Cheu-Wu et al., 1988), S. cerevisiae a' (CKA2;Padmanabha et al., 1990), C. elegans (C. elg, Hu & Rubin, 1990), D. melanogaster (D. mel.; Saxena et al., 1987). The numbering is according to that of Drosophila CK I1 a subunit (Saxena et al., 1987; Padmanabha et al., 1990). Arrowheads indicate residues present in virtually all kinases (Hanks & Quinn, 1991). The boxed residues are those present in all CK I1 catalytic subunits compared here. The total length, in amino acid residues, of each CK I1 catalytic subunit is given at the end of each sequence.
seen in other genes of T.parva (R. Bishop, personal communication). There were two sequences, starting at positions 1343 and 1355 (ATTTA and TTTA, respectively) in the 3' untranslated region, which have been reported to confer instability to mRNA (Shaw & Kamen, 1986). All three of these sequence motifs were present in the cDNA clones with sequence identity spanning nucleotides 1 138-1 374. Within the T. parva CK I1 a! gene were two domains (nucleotides 800-900 and 1001-1 100) within which the gene had greater than 71% identity with the gene for the Drosophila CK I1 cy subunit (Saxena et al., 1987). Contained within the latter domain was a 37-bp region for which the two sequences are nearly 90% identical. When the Drosophila CK I1 cDNA a! gene was used to probe HpaII-digested XgTpMCK2 DNA, we had noted that the enzyme cut the insert fragments of about 1.4 and 0.9 kb that hybridized to the heterologous probe, giving signals of equal intensity (data not shown). The HpaII cutting site, which lies between these two conserved nucleotide sequences at nucleotide 935, is consistent with those observations. Interestingly, this HpaII site is within the part of the gene encoding the conserved KGPELLV sequence motif, which is present in all CK I1 a! subunits reported to date.
The T.parva CK I1 a! gene appears to have no introns and potentially encodes a protein larger than most other CK I1 a! subunits described in other species. For confirmation of transcription of this gene, we obtained sequence information from a cDNA clone. This clone had an insert of 1.5 kb and revealed complete identity with the consensus genomic sequence from base 53 to 1374. Comparison of Deduced Amino Acid Sequences of CK 11 a! Subunits from Invertebrates. The amino acid sequence deduced from the T.parva casein kinase I1 a! subunit sequence has a high degree of identity with the cell division control (cdc) kinases, as has been reported for CK I1 a! sequences from other organisms (Lorincz & Reed, 1984; Takio et al., 1987; Saxena et al., 1987; Chen-Wu et al., 1988). Pairwise comparisons between the T.parva CK I1 a! sequence, with identity generally beginning after the conserved tyrosine (Y3' in the D. melanogaster CK I1 a! sequence; Figure 3), with the cdc2 human analogue, cdc28, and cdc2 gives identities of 31%, 30%, and 29%, respectively. The alignment of the predicted T.parva CK I1 a! subunit sequence with those from other invertebrates (S. cerevisiae, C. elegans, and D. melanogaster) is shown in Figure 3. As has been reported for other catalytic subunits
Theileria parva Casein Kinase I1 a! Subunit of CK 11, the T. parva sequence has Ser instead of Gly in the sequence motif G4XGXXSXV in catalytic subdomain I, Val instead of Ala in the A@XKsequence in catalytic subdomain 11, DW175Ginstead of DF175Gin subdomain VII, and G195PE instead of A195PEin subdomain VIII. The last two replacements appear to be unique to the catalytic subunits of casein kinase I1 (Padmanabha et al., 1990). The boxed residues shown in Figure 3 are the amino acids that are present in all the five catalytic subunits compared here. In general these CK I1 a! subunits from lower eukaryotes do not differ substantially from a wider comparison in an alignment which included CK I1 catalytic subunits from mammalian sources (Padmanabha et al., 1990). Of the residues shown to be conserved in all casein kinase I1 catalytic subunits reported by Padmanabha et al. (1990), there are 17 amino acids which are different in the T. parva sequence reported here. Among the 17 differences, however, only one (V304/R) represents a radical replacement. The T. parva CK I1 subunit displays 68% sequence identity with the Drosophila a! subunit, 67% with the C. elegans a! subunit, 58% with the S. cerevisiae d subunit, and 56% with the S. cerevisiae a! subunit. Among mammalian CK I1 catalytic subunits, the predicted T. parva sequence has about 68% sequence identity with the human, rat, and chicken a! subunits (Lozeman et al., 1990; Meisner et al., 1989; Maridor et al., 1991), while identity with the a!’ subunits from these species was less, ranging from 65% to 66%. Casein kinase I1 catalytic subunits have thus been remarkably conserved across wide evolutionary distances. Padmanabha et al. (1990) pointed out three domains of interest in the casein kinase I1 subfamily. These include the ARVY and EYWDYE sequence motifs which lie N-terminal to the kinase catalytic subdomain I (Hanks & Quinn, 1991) and the KPVKKKKIKR sequence within catalytic subdomain 11. The latter two are completely conserved in T. parva CK I1 a! sequence. However, the T. parva sequence PKTY, replacing ARVY, contains three conservativereplacements, thus leaving tyrosine as the only invariant residue in this sequence motif. With the exception of the S. cerevisiae CK I1 a! subunit (CKAI),potential N-linked glycosylationsites (NXS/T) were present in all the CK I1 sequences compared. Among the CK I1 catalytic subunit sequences available, the T. parva subunit has the highest number ( 5 ) of potential N-linked glycosylation sites. Only one of these (NNT/S; subdomain V) is shared with the other sequences, including those from mammalian sources. With this one exception, these sites fall within sequence stretches for which the T. parva CK I1 catalytic subunit has no homology with all the other a! or CY’ subunits. A Novel N-Terminal Extension on T. parva CK II. The T.parva CK I1 catalytic subunit has a unique N-terminal99 amino acid sequence. Visual inspection of this part of the sequence revealed a putative signal peptide (residues 1-26). We then employed the probability weight-matrix algorithm described by von Heijne (1986) to identify putative signal sequences and their probable peptidase cleavage sites within the whole of the deduced T. parva CK I1 a! sequence. There were five frames that had marginally high Si values (von Heijne, 1986), two of which were within the N-terminal sequence unique to T. parva. These sequences consisted of a hydrophobic stretch of amino acids, the h-region (residues 10-19), bounded on the N-terminal side by a group of amino acids with a net positive charge, the n-region (residues 1-9), and on the C-terminal end by a stretch of residues, the c-region (residues 20-26), which are more polar than the hydrophobic core.
Biochemistry, Vol. 31, No. 27, 1992 6199 1 2 3 4 5 6 7 8
23.09.4-
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0.60.3FIGURE 4: Genomic Southern blot analysis of T.purvu piroplasm DNA using the EcoO109I/XhoIprobe. Total T.purvu genomic DNA was digested with each of various restriction enzymes, and the fragments were resolved on an (0.8%) agarose gel and blotted onto a nylon filter. The enzymes included BumHI (lane l), EcoRI (lane 2), Hind111 (lane 3), AseI (lane 4),EcoO109I/XhoI(lane 5), KpnI (lane 6 ) , PstI (lane 7), and StuI (lane 8). The conditions for hybridization and washing are as described under Materials and Methods. Autoradiography was overnight at -70 O C with intensifying screens. The DNA fragment length markers are given in kilobases.
Southern Blot Analysis of the T. parva CK 11 Gene. Genomic DNA from T. parva piroplasms was digested to completion with various restriction enzymes. An EcoO109I/XhoI fragment (residues 395-1 282, which is contained entirely within the T. parva CK I1 a! open reading frame) was radiolabeled and used to probe Southern blots of these digests (Figure 4). The restriction enzymes, which have no sites within the sequenced T. parva CK I1 gene, generated DNA fragments larger than the gene [BamHI (8.0 kb), EcoRI (15.0 kb), HindIII (10 kb), KpnI (8.3 kb), PstI (15 kb), and StuI (15 kb)]. The EcoRI and HindIII fragments, which hybridized to the labeled Eco0109I/XhoI probe, are the same lengths as those seen earlier when T.parva digests were probed with the Drosophila CK I1 a! subunit probe. Enzymes with restriction sites within the CK I1 a! gene generated fragments of expected lengths. For example, AseI, with cutting sites at nucleotides 164, 1398, and 1527, generated one large strongly hybridizing fragment of about 1.25 kb (Figure 4, lane 4). The other fragment showed no hybridization because it was outside the sequence complementary to the probe. Use of restriction endonucleases having two or more sites within the gene, including TaqI, HpaII, Sad, and NciI (data not shown), revealed, in each case, the expected number of fragments hybridizing to the EcoO109I/XhoI probe. Although these data are not completely conclusive, they suggest that T. parva has a single copy of the CK I1 a gene. Figure 4 also shows that in addition to the main band, all of the other restriction enzymes, perhaps with the exception of EcoRI (Figure 4, lane 2) and PstI (Figure 4, lane 7), generated a weakly hybridizing band, which is best seen in the AseI digest (Figure 4, lane 4) where it appears as a satellite above the main band. T. parva CK 11 Gene Transcripts. Transcripts derived from the T. parva CK I1 gene were analyzed by northern blot hybridization of poly(A)-enriched RNA prepared from the IL 2-dependent G6 clone, the G6TpM T. parva-infected cell line, and T. parva piroplasms isolated from blood of infected cattle. An ethidum bromide-stained gel (Figure 5, lanes 1-3) and an autoradiogram of the northern blot are shown. The T. parva CK I1 a! Riboprobe showed no hybridization to RNA
6200 Biochemistry, Vol. 31, No. 27, 1992 A
B
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5: Northern blot analysis of bovine and T. puma poly(A)-enriched RNA using, as a probe, the 888-bp Eco0109I/XhoI fragment of the T. puma CK I1 a gene, which was labeled with 32P. Poly(A)-enriched RNA was prepared from the IL 2-dependent G6 T-lymphocyte clone, from the same clone (G6TpM) after it had been infected with T . parva, and from T. pama piroplasms. The poly(A)-enriched RNAs were resolved in an agarose-formaldehyde gel and blotted onto a filter. The ethidium bromide-stained gel is shown in panel A. Lanes 1 and 2 contained 10 pg each of poly(A) RNA from the uninfected IL 2-dependent G6 parental cell line and the G6TpM cell line infected with T. pama (Muguga), respectively. Poly(A)-rich RNA (1 pg) from piroplasms of T. parua is shown in lane 3. The filter was hybridized with a 32P-labeledantisense Riboprobe synthesized in vitro using a 1.5-kb T.puma CK I1 a cDNA as described under Materials and Methods. The amounts of T. purvu poly(A)-enriched RNA used in this experiment were estimated by assuming that 10% of the poly(A)-rich RNA isolated from infected lymphocytes is of theileria1 origin as estimated by Rot analysis (Gerhards et al., 1989). The filter was washed with 0.5X SSC/O.l% SDS for 1 h and exposed to X-ray film at -80 “C for 1 day. The size markers are the RNA ladder from BRL. FIGURE
from the uninfected G6 cell line. In contrast, the CK I1 a! probe hybridized with RNA from the uninfected G6 cell line. In contrast, the CK I1 a! probe hybridized with RNA from the G6TpM T-cell clone infected with T. purvu as well as to piroplasm RNA, giving bands of equal intensity at 1.65 kb. There was therefore no detectable length heterogeneity in the T. pama CK I1 a! transcripts, as has been reported for higher eukaryotes (Meisner et al., 1989; Litchfield et al., 1990; Maridor et al., 1991).
DISCUSSION In this study, we describe the cloning and characterization of a gene for the casein kinase I1 a! subunit of the intracellular, bovid lymphocyte-transformingprotozoan parasite Theileria purvu. The deduced amino acid sequence for the T. purvu enzyme has the amino acid residues which are conserved in virtually all protein kinases, as well as those specific for the serine/threonine kinase family (Padmanabha et al., 1990; Hanks & Quinn, 1991). With the exception of the ARVY sequence motif located near the N-terminus of all the other casein kinase I1 a! or a!’ subunits, the T. purvu enzyme has essentially all the structural features considered unique to the catalytic subunits of casein kinase I1 (Saxena et al., 1987; Chen-Wu et al., 1988; Padmanabha et al., 1990). These include the EYWDYE, the basic sequence motif KPVKKKKIKR, DWG, and GPE. The sequence differences between CK I1 and other kinases, even though some involve conservative replacements when compared to those of other kinase subfamilies, may be sufficient to confer the functional
ole-MoiYoi et al. characteristics unique to CK 11, such as the capacity to utilize GTP, as well as ATP, as a phosphoryl group donor and its susceptibilityto modulation by a variety of effector molecules, including its exquisite sensitivity to inhibition by heparin and other glycosaminoglycans (Feige et al., 1980; Hathaway et al., 1980). In an attempt to determine whether the gene that we have cloned encoded the a! or the d subunit of CK 11, we performed a protein sequence comparison between the T.purvu catalytic subunit and the CK I1 a! and a!’ sequences from human (Lozman et al., 1990) and chicken (Maridor et al., 1991). This comparison revealed that, among the positions for which the a! and d sequences differed, there were 18 residues for which the T. purvu catalytic subunit was identical with either the a! or the d sequences from the other two species. Among these residues, the T. purvu sequence was most identical with the a! (13 of 18 residues) rather than the d subunit. The T. parvu gene that we have cloned is, therefore, likely to be that encoding the CK I1 a! subunit of the enzyme. A most intriguing feature of the T.purvu CK I1 a! subunit is the presence of a deduced N-terminal99 amino acid peptide. Within this region of the molecule is a sequence, residues 1-26 of the T. purvu CK I1 a! sequence, which has features characteristic of presecretory signal peptides (von Heijne, 1986; Gierasch, 1989). These features include the presence of the three signal sequence regions reported by von Heijne (1986): an n-region, which carries a net positive charge, an h-region, consisting of a hydrophobic core, and a c-region, with one or more polar residues. In addition to this putative signal peptide, there are five potential N-linked glycosylation sites in the predicted sequence of the T. purvu CK I1 a! subunit, four of which are not present in any of the other CK I1 catalytic subunits. If these sites are at all glycosylated, it would be an additional indicator that the T.purvu CK I1 is either secreted or destined for the plasma membrane. We have not found reports in the literature of a CK I1 with such structural features. However, other serine/threonine kinases have been detected in a variety of mammalian plasma membrane preparations and in intact cells including transformed 3T3 cells, adipocytes, HeLa cells, macrophages, mouse lymphoma cells, and myoblasts (Mastro & Rozengurt, 1976; Remold-O’Donnell, 1978; Kubler et al., 1989; Chen & Lo, 1991). Such ectoprotein kinases phosphorylate endogenous cell membrane proteins as well as those present in the extracellular milieu (Chen & Lo, 1991). It has been suggested that the highly basic sequence motif (KPVKKKKIKR) may mediate interaction of the a! subunit with the highly acidic domains of the @ subunit as well as with those of protein substrates, most of which are acidic, and with a variety of effector molecules [Chen-Wu et al., 1988; Lozeman et al., 1990; see review by Tuazon and Traugh (1991)l. It has been noted by Lozeman et al. (1990) that the KPVKKKKIKR sequence also has the features characteristic of nuclear localization sequence (NLS) motifs (Kalderon et al., 1984; Lanford & Butel, 1984; Richardson et al., 1986; Dang & Lee, 1989). NLS motifs have been shown to be present in about a dozen nuclear proteins phosphorylated by CK I1 and are located at an average distance of about 10-30 amino acid residues N-terminal to putative CK I1 phosphorylation sites (Rihs et al., 1991). Rihs and Peters (1989) have shown that the NLS motif of SV40 large T antigen was sufficient to induce nuclear accumulation of recombinant fusion proteins containing large T antigen. It is of considerable interest that Rihs et al. (1991) have recently shown that, whereas the specificity of nuclear targeting of proteins is de-
Biochemistry, Vol. 31, No. 27, 1992 6201
Theileria parva Casein Kinase I1 a Subunit termined by the N U motif, the rate of transport of the SV40 large T antigen was a function of phosphorylation of the CK I1 site in the vicinity of the N U . It has recently been proposed that the dynamics of cytoplasmic/nuclear translocation of molecules capable of modulating cellular replication represents an important level of regulation of cellular growth and differentiation (Rihs et al., 1991). In this context, the possible role of CK I1 as a transducer of growth signals to the nucleus might be effected through the alteration of the rate of nuclear protein transport, a process which would clearly influence gene expression and cell growth. That the CK I1 a’ subunit is present in the nucleus and, like the CK I1 a subunit, shows a change in distribution depending on the stage of the cell cycle, raises questions regarding differences in the functions of the catalytic subunits of CK 11. Chen-Wu et al. (1988)and Padmanabha et al. (1990) showed that disruption of either the a or the a’subunit genes of S . cerevisiae casein kinase I1 was not lethal and concluded that the catalytic subunits are sufficiently similar to allow each subunit to compensate for the absent functions of the other. However, haploid yeast cells with mutations of both catalytic subunits were growth-arrested and nonviable. Padmanabha et al. (1990)could rescue such yeast cells with double mutations of the catalytic subunits by complementation with the Drosophila CK I1 a subunit; these observations provide functional evidence reflecting the remarkable degree of primary structural conservation of CK I1 over great evolutionary distances. In addition to identifying the gene encoding the T. parva CK I1 a subunit, we have also detected casein kinase I1 activity in lysates of schizonts purified from T. parva-infected bovine lymphocytes. However, we have not shown that the casein kinase I1 gene encodes the enzyme whose activity we have detected. It is known that CK I1 aggregates to form insoluble, linear filaments in buffers of low ionic strength (Glover, 1986). Although the buffers used to purify schizonts were of sufficiently high ionic strength (Sugimoto et al., 1988) to prevent such precipitation and subsequent copurification of bovine CK I1 with the schizonts, this possibility has not been entirely ruled out. In conclusion, the structure of the a subunit of casein kinase I1 from T. parva adds information to recent observations showing that CK I1 is extremely well conserved among highly divergent species. Differences in molecular masses between the a and the a’ subunits of CK I1 are attributable to Cterminal extensions in the case of vertebrates (Lozeman et al., 1990;Meisner et al., 1989;Maridor et al., 1991) or an internal insertion in the case of yeast (Chen-Wu et al., 1988;Padmanabha et al., 1990). The CK I1 a subunit of T. parva, reported here, has a large N-terminal extension. Evidence has been presented by others to show that CK I1 is activated upon hormonal or growth factor stimulation of various cell types (Sommercorn & Krebs, 1987;Sommercorn et al., 1987;Klarlund & Czech, 1988;Krebs et al., 1988; Carroll & Marshak, 1988;Ackerman & Osheroff, 1989). Additionally, increased CK I1 activity has been observed in rapidly proliferating cells such as human leukemic and virally-transformed cell lines [reviewed by Pinna (1990) and Tuazon and Traugh (1991)l. In this study, we have not established a connection between the T. patva CK I1 a and Theileria-induced lymphocyte transformation. However, T . parva schizonts are completely exposed to the host cell’s cytoplasm. They may therefore induce cellular transformation by releasing effector molecules into the lymphocyte, which then activate pathways for cellular proliferation. Such molecules may also be exposed on the schizont’s plasma membrane. In
either of these cases, an essential feature of such parasite molecules would be their capacity to effectively interact with, and modulate, host substrates, which implies interesting structural as well as functional conservation of the molecules from both species. The T. pama CK I1 a subunit has a high degree of identity with the catalytic subunits of CK I1 from higher eukaryotes and has structural features which accord with those of molecules targeted for translocation to the nucleus as well as for secretion. ACKNOWLEDGMENTS We are grateful to Dr. Claiborne V. C. Glover (University of Georgia, Athens, GA) for Drosophila cDNA clones and for his interest, helpful discussions, and provision of manuscripts prior to publication, to Dr. Michael E. and Grace K. Dahmus (University of California, Davis, CA) and to Drs. Daniel R. Marshak and I1 Je Yu (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) for their helpful discussions and for provision of copies of manuscripts prior to publication. We acknowledge Dr. Paul T. Englund and our colleagues at ILRAD, R. P. Bishop, W. R. Fish, J. D. Lonsdale-Eccles, N. B. Murphy, and D. Zilberstein, for critical comments on the manuscript and Mrs. L. W. Thairo for typing the manuscript. REFERENCES Ackerman, P., & Osheroff, N. (1989) J . Biol. Chem. 264,
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